Distinct kinetics of human DNA ligases I, IIIα, IIIβ, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair
Distinct kinetics of human DNA ligases I, IIIα, IIIβ, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair
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DOI：
10.1016/j.dnarep.2009.06.002
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发表时间：
2009-08-06
期刊：
影响因子：
3.8
通讯作者：
Wilson, Gerald M.
中科院分区：
文献类型：
--
作者：
Chen, Xi;Ballin, Jeff D.;Wilson, Gerald M.
这三个人类LIG基因编码的多肽在DNA复制、重组和修复过程中催化磷酸二酯键的形成。虽然许多研究已经确定了人类DNA连接酶（hLigs）的蛋白质伴侣，但对这些酶的催化特性的表征却很少。在这项研究中，我们开发并优化了一种基于荧光的DNA连接试验，以表征纯化的高ligs的活性。虽然hLigI加入DNA缺口，但它在具有短内聚单链末端的线性双链DNA底物上没有可检测到的活性。相比之下，hLigIII β和hLigIII α /XRCC1和hLigIV/XRCC4复合物在缺口和线性双工DNA底物上都有活性。令人惊讶的是，作为主要非同源末端连接（NHEJ）途径的关键组分，hLigIV/XRCC4在线性双工DNA底物上的活性明显低于hLigIII。值得注意的是，hLigIV/XRCC4分子仅在ATP缺失或存在的情况下催化单个连接事件。未能催化后续的连接事件反映了下一个连接反应的酶-腺苷化步骤的缺陷，并表明，除非存在一种体内机制来在磷酸二酯键形成后重新激活DNA连接酶IV/XRCC4，否则细胞的NHEJ容量将由腺苷化的DNA连接酶eiv /XRCC4分子的数量决定。(C) 2009 Elsevier B.V.版权所有
The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic Properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIII beta and the hLigIII alpha/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules. (C) 2009 Elsevier B.V. All rights reserved.